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Research Project: Innovations that Improve the Efficiency and Effectiveness of Managing and Preserving Ex Situ Plant Germplasm Collections


2016 Annual Report

Genebanks have an urgent mandate to increase efficiency and number of germplasm forms and species within collections. This mandate will be achieved through technologies that prolong germplasm shelf-life, tools that nondestructively detect early changes in viability and genetic integrity, and methods that quantify and compare wild and collected diversity. Through time, genebanked materials must stay fit-for-purpose. PGPRU’s goals are to provide state-of-art genebanking methods that address current needs of genebank operators to manage collections cost-effectively and future needs of users for access to well characterized, diverse collections of living germplasm. To meet this challenge, PGPRU will perform research in the next 5 years that will: OBJECTIVE 1: DETECT GAPS AND REDUNDANCIES IN GENEBANK COLLECTIONS Develop and validate methods that integrate statistical genetics and spatial analyses to detect gaps and redundancies in genebank collections, and to estimate and compare the genetic diversity among genebank collections and in situ populations, especially for crop landraces and wild relatives. • Subobjective 1a. Partition genetic diversity within NPGS collections into subsets and provide metrics to relate genetic distance among accessions. • Subobjective 1b. Confirm whether geospatial and climatic data can identify collection gaps or ecotypes. OBJECTIVE 2: IMPROVE INITIAL AND LONG-TERM SURVIVAL OF STORED GERMPLASM Devise or refine tools that enhance the long-term viability of stored germplasm, including clonal propagules, and provide the means for curators to assess and predict the response of germplasm to conventional and cryogenic storage treatments. • Subobjective 2a: Develop methods to recover vigorous plants from shoot tips. • Subobjective 2b: Quantify variation of shoot tip response to established preservation methods of desiccation and liquid nitrogen exposure. • Subobjective 2c: Quantify variation of dormant bud response to preservation methods of desiccation and liquid nitrogen exposure. • Subobjective 2d: Quantify variation of seed response to desiccation and cooling. • Subobjective 2e: Quantify interactions between temperature, moisture and seed longevity. OBJECTIVE 3: EVALUATE CHANGES IN QUALITY AND GENETIC IDENTITY Design metrics for monitoring and validating biological quality (viability, health, etc.) of stored and regenerated plant germplasm and assess genetic integrity of germplasm and the genetic shifts that occur during germplasm management. • Subobjective 3a: Develop new tools to measure initial vigor and detect aging. • Subobjective 3b: Assess risks of genetic change during genebanking. OBJECTIVE 4: LOCATE MASKED DESIRABLE GENES IN CROP WILD RELATIVES Develop and apply genome annotation methods to evaluate collections of crop land races and wild relatives for genetic diversity in key agricultural traits, so as to enable more effective germplasm curation and to improve access to that diversity for marker-assisted breeding.

The Plant Germplasm Preservation Research Unit (PGPRU) has the unique mission of troubleshooting plant genebanking methods to solve the most critical problems of genetic resource collections: keeping germplasm alive, healthy and representative of the source population; describing collection composition; and ensuring stored germplasm meets the needs of diverse users. This 5 year plan describes PGPRU’s strategy to apply creative, multidisciplinary approaches that balance the special requirements of diverse living germplasm (seeds, pollen and explants) with the practical needs of curators and users. Research will provide tools that compare genetic diversity of collections with species diversity extant in the wild; broaden the array and longevity of propagules in storage; assess germplasm health with minimum sample depletion; and account for genebanking effects on the biological and genetic integrity of the sample. Our research efforts will be vital to the overall goal of creating relevant scientific collections to understand, protect and use plant diversity in a changing world. PGPRU will approach the task of improving genebanking as the challenge of achieving apparently contradictory goals, such as maximizing genetic diversity while minimizing collection size; standardizing preservation treatments for diverse propagules that respond differently; monitoring for signs of deterioration during early storage when few changes are known to occur; maintaining genetic heterogeneity in an agricultural context where quality and uniformity are highly valued; and finding specific alleles of interest that may be masked by the genetic background. In a real sense, these contradictions underscore the complex mission of genebanking. PGPRU will remain at the forefront of plant repository biology and will continue to play a global role in technology transfer for plant genebank management.

Progress Report
Objectives 1 and 4: Much of the Plant Germplasm Preservation Research Unit (PGPRU) efforts in these objectives have evolved towards using large data sets of genomic or environmental data to characterize genetic resource collections and inform collection management practices. Management decisions addressed this year include: identifying core subsets that maximize diversity with minimum accession numbers, selecting parent trees in clonal repositories for controlled pollinations for seed collections (easier to store as backups than vegetative propagules), prioritizing accessions that are not already backed up at NCGRP, comparing genetic diversity in current collections with historical levels of genetic diversity, locating genetic diversity in the wild that maybe useful but is not already included within NPGS collections, and characterizing genetic changes of wild populations in response to climate change. PGPRU scientists are actively engaged with the international community to develop methods to use genomic and environmental data to guide collections of crop wild relatives (CWR) and find the useful genes. Objective 2: Major advances in our abilities to cryopreserve germplasm are exemplified for diverse species and tissues. Probably the most impactful progress is the technology to rapidly process citrus shoot tips to ensure back-up of the collection in Riverside, CA. In addition to this, progress is being made on a number of fronts including grape, recalcitrant seeds from trees and seeds with physiology intermediate to recalcitrant and orthodox seeds. Hence the number of species that can be included in NCGRP’s cryopreserved collections is increasing dramatically. Most of these successes arise from fundamental understanding of the preservation process and the expression of damage and recovery of cells when they transition between fluid (living) and solid (preserved) states. Collaborative work with the PAGRP also progressed towards a decision tool to process and aliquot propagules based on the probability of obtaining the needed number of viable propagules. This new tool will be incorporated into SOPs for handling clonally propagated germplasm. Objective 3: This objective is designed to evaluate changes to germplasm as a result of the genebanking process. Progress has been made in developing better theory to sample populations in the wild to ensure they represent the extant genetic diversity. Within the genebank, there has been substantial progress in developing tools to evaluate changed quality of stored germplasm. One such tool uses dynamic mechanical analysis (DMA) in a comparable way as materials engineers, who predict stability properties of plastics. Another tool that quantifies changes in RNA integrity with storage time is the first instance where a chemical constituent correlates closely with seed viability and leads to the possibility of a surrogate for germination assays. All the genotyping and phenotyping work has been completed on a large international project investigating the incidence of genetic drift or erosion that occurs in stored seeds of wild populations of barley. This information will serve to parameterize models to simulate genetic change in genebanks.

1. Technologies to efficiently cryopreserve citrus germplasm. Highly reliable methods to cryopreserve shoot tips of citrus have been developed and are currently being used in an accelerated initiative to back up the citrus genetic resource collections in Riverside, California. In an “all hands on deck” effort, three research units are collaborating to secure 478 high priority Citrus accessions in a year. One third of the entire citrus collection will be securely backed up within the year.

2. Tools that improve efficiency within genebanks. A number of tools have been developed and are available for implementation to increase efficiencies of plant genetic resource collections. These tools include various methods to partition large collections into core subsets, molecular approaches to identify seed parents that can capture and maintain genetic diversity of trees; probability functions that estimate number of viable propagules in cryopreserved collections and RNA integrity assays that track and possibly supplant destructive germination testing. Genebanks can represent genetic diversity with fewer samples that take less processing time and viability monitoring will not deplete the collection.


Review Publications
Hardegree, S.P., Walters, C.T., Boehm, A.R., Olsoy, P.J., Clark, P., Pierson Jr, F.B. 2015. Hydrothermal germination models: comparison of two data-fitting approaches with probit optimization. Crop Science. 55:2276-2290. doi:10.2135/cropsci2014.10.0703.
Volk, G.M., Henk, A.D. 2016. Historic American apple cultivars: Identification and availability. Journal of the American Society for Horticultural Science. 141(3):292-301.
Li, B., Feng, C., Hu, L., Wang, M., Volk, G.M., Wang, Q. 2015. Recovery patterns, histological observations and genetic integrity in Malus shoot tips cryopreserved using droplet vitrification and encapsulation-dehydration procedures. Journal of Biotechnology. 214:182-191.
Zhang, J., Huang, B., Zhang, X., Volk, G.M., Xin, X., Yin, G., He, J., Lu, X., Zhou, Y., Chen, X. 2015. Identification of a highly successful cryopreservation method (droplet-vitrification) for petunia. In Vitro Cellular and Developmental Biology - Plants. 51:445-451.
Roose, M. L., Gmitter F. G., Lee, R.F., Hummer, K.E., Machado, M., Ashmore, S., Deng, X., Ancillo, G., Vives, M.C., Volk, G.M., Kahn, T.L., Luro, F. 2015. Development of a global conservation strategy for citrus genetic resources. Acta Horticulture Proceedings. 1065:75-83.
Gao, Y., Liu, F., Wang, K., Wang, D., Gong, X., Liu, L., Richards, C.M., Henk, A.D., Volk, G.M. 2015. Genetic diversity of Malus cultivars and wild relatives in the Chinese National Repository of Apple Germplasm Resources. Tree Genetics and Genomes. 11:106. doi:10.1007/s11295-015-0913-7.
Mira, S., Hill, L.M., Gonzalez-Benito, M., Ibanez, M.A., Walters, C.T. 2016. Volatile emission in dry seeds as a way to probe chemical reactions during initial asymptomatic deterioration. Journal of Experimental Botany. 67(6):1783-1793.
Hufbauer, R.A., Szucs, M., Kasyon, E., Youngberg, C., Koontz, M.J., Richards, C.M., Tuff, T., Melbourne, B.A. 2015. Three types of rescue can avert extinction in a changing environment. Proceedings of the National Academy of Sciences. 112(33):10557-10562.
Xia, K., Hill, L.M., Decorte, A., Li, D., Walters, C.T. 2014. Factors affecting stress tolerance in recalcitrant embryonic axes from four Quercus (Fagaceae) species native to the US or China. Annals Of Botany. 114(8):1747-1759.